scholarly journals Single channel studies of the phosphorylation of K+ channels in the squid giant axon. I. Steady-state conditions.

1991 ◽  
Vol 98 (1) ◽  
pp. 1-17 ◽  
Author(s):  
E Perozo ◽  
C A Vandenberg ◽  
D S Jong ◽  
F Bezanilla
Keyword(s):  
Steady State ◽  
Single Channel ◽  
Slow Component ◽  
Giant Axon ◽  
Delayed Rectifier ◽  
Open Probability ◽  
Delayed Rectifier Current ◽  
K Current ◽  
The Difference ◽  
Single Channel Currents

Phosphorylation of the delayed rectifier channel of squid potentiates the macroscopic K+ current and slows its activation kinetics. We have studied this phenomenon at the single channel level using the cut-open axon technique under steady-state conditions. In 10 mM external K+/310 mM internal K+ there are predominantly two types of channels present, a 20-pS and a 40-pS channel. In steady state at depolarized potentials, the 40-pS channel was most active, whereas the 20-pS channel tended to disappear due to a slow inactivation process. Two methods were developed to shift the population of channels toward a dephosphorylated state. One method consisted of predialyzing a whole axon with solutions containing no ATP, while recording the currents under axial-wire voltage clamp. A piece of axon was then removed and cut open, and single channel currents were recorded from the cut-open axon. A second method was based on the difference in diffusion coefficients for ATP and proteins such as the endogenous phosphatase. The axon was cut open in a solution that did not contain Ca2+ or Cl- in order to maintain the axoplasm structurally intact and permit endogenous phosphatase to act on the membrane while ATP diffused away, before removing the axoplasm and forming a membrane patch. When dephosphorylating conditions were used, the steady-state open probability of the 40-pS channel at 42 mV was very low (less than 0.0002), and the channel openings appeared as a series of infrequent, short-duration events. The channel activity was increased up to 150-fold by photoreleasing caged ATP inside the patch pipette in the presence of the catalytic subunit of protein kinase A. The sharp increase in open probability could be accounted for by a decrease of the slow component of the closed time distribution from 23 s to 170 ms with little change in the distribution of open times (1-2 ms) and no change in the single channel current amplitude. In voltage-jump experiments the contribution of the 40-pS channel to the delayed rectifier current was often small due to the large values of the latency to the first opening.

scholarly journals Charybdotoxin selectively blocks small Ca-activated K channels in Aplysia neurons.

1987 ◽  
Vol 90 (1) ◽  
pp. 27-47 ◽  
Author(s):  
A Hermann ◽  
C Erxleben
Keyword(s):  
Single Channel ◽  
Delayed Rectifier ◽  
Pacemaker Activity ◽  
Dependent Manner ◽  
Open Probability ◽  
Apparent Dissociation Constant ◽  
External Application ◽  
K Channels ◽  
Voltage Dependent ◽  
K Current

The action of charybdotoxin (ChTX), a peptide component isolated from the venom of the scorpion Leiurus quinquestriatus, was investigated on membrane currents of identified neurons from the marine mollusk, Aplysia californica. Macroscopic current recordings showed that the external application of ChTX blocks the Ca-activated K current in a dose- and voltage-dependent manner. The apparent dissociation constant is 30 nM at V = -30 mV and increases e-fold for a +50- to +70-mV change in membrane potential, which indicates that the toxin molecule is sensitive to approximately 35% of the transmembrane electric field. The toxin is bound to the receptor with a 1:1 stoichiometry and its effect is reversible after washout. The toxin also suppresses the membrane leakage conductance and a resting K conductance activated by internal Ca ions. The toxin has no significant effect on the inward Na or Ca currents, the transient K current, or the delayed rectifier K current. Records from Ca-activated K channels revealed a single channel conductance of 35 +/- 5 pS at V = 0 mV in asymmetrical K solution. The channel open probability increased with the internal Ca concentration and with membrane voltage. The K channels were blocked by submillimolar concentrations of tetraethylammonium ions and by nanomolar concentrations of ChTX, but were not blocked by 4-aminopyridine if applied externally on outside-out patches. From the effects of ChTX on K current and on bursting pacemaker activity, it is concluded that the termination of bursts is in part controlled by a Ca-activated K conductance.

scholarly journals Single-channel currents from diethylpyrocarbonate-modified NMDA receptors in cultured rat brain cortical neurons.

1995 ◽  
Vol 105 (6) ◽  
pp. 837-859 ◽  
Author(s):  
J L Donnelly ◽  
B S Pallotta
Keyword(s):  
Steady State ◽  
Rat Brain ◽  
Cortical Neurons ◽  
Single Channel ◽  
Channel Gating ◽  
Reversal Potential ◽  
Open Probability ◽  
Histidine Residues ◽  
Gating Model ◽  
Single Channel Currents

The role of histidine residues in the function of N-methyl-D-aspartate (NMDA)-activated channels was tested with the histidine-modifying reagent diethylpyrocarbonate (DEP) applied to cells and membrane patches from rat brain cortical neurons in culture. Channels in excised outside-out patches that were treated with 3 mM DEP for 15-30 s (pH 6.5) showed an average 3.4-fold potentiation in steady state open probability when exposed to NMDA and glycine. Analysis of the underlying alterations in channel gating revealed no changes in the numbers of kinetic states: distributions of open intervals were fitted with three exponential components, and four components described the shut intervals, in both control and DEP-modified channels. However, the distribution of shut intervals was obviously different after DEP treatment, consistent with the single-channel current record. After modification, the proportion of long shut states was decreased while the time constants were largely unaffected. Burst kinetics reflected these effects with an increase in the average number of openings/burst from 1.5 (control) to 2.2 (DEP), and a decrease in the average interburst interval from 54.1 to 38.2 ms. These effects were most likely due to histidine modification because other reagents (n-acetylimidazole and 2,4,6-trinitrobenzene 1-sulfonic acid) that are specific for residues other than histidine failed to reproduce the effects of DEP, whereas hydroxylamine could restore channel open probability to control levels. In contrast to these effects on channel gating, DEP had no effect on average single-channel conductance or reversal potential under bi-ionic (Na+:Cs+) conditions. Inhibition by zinc was also unaffected by DEP. We propose a channel gating model in which transitions between single- and multi-opening burst modes give rise to the channel activity observed under steady state conditions. When adjusted to account for the effects of DEP, this model suggests that one or more extracellular histidine residues involved in channel gating are associated with a single kinetic state.

scholarly journals Single channel studies of the phosphorylation of K+ channels in the squid giant axon. II. Nonstationary conditions.

1991 ◽  
Vol 98 (1) ◽  
pp. 19-34 ◽  
Author(s):  
E Perozo ◽  
D S Jong ◽  
F Bezanilla
Keyword(s):  
Voltage Dependence ◽  
Single Channel ◽  
Giant Axon ◽  
Squid Giant Axon ◽  
Delayed Rectifier ◽  
Activation Process ◽  
Inactivation Curve ◽  
Caged Atp ◽  
Delayed Rectifier Current ◽  
Voltage Curve

The effects of phosphorylation on the properties of the 20-pS channel of the squid giant axon were studied using the cut-open axon technique. Phosphorylation of the channel was achieved by photoreleasing caged ATP (inside the patch pipette) in the presence of the catalytic subunit of the protein kinase A. An inverted K+ gradient (500 K+ external parallel 5 K+ internal) was used to study the activation process. Phosphorylation decreased the frequency of openings of the channel at most potentials by shifting the probability vs. voltage curve toward more positive potentials. The mean open times showed no voltage dependence and were not affected by phosphorylation. The distribution of first latencies, on the other hand, displayed a sharp voltage dependence. Phosphorylation increased the latency to the first opening at all potentials, shifting the median first latency vs. voltage curve toward more positive potentials. The slow inactivation process was studied in the presence of a physiological K+ gradient (10 K+ external parallel 310 K+ internal). Pulses to 40 mV from different holding potentials were analyzed. Phosphorylation increases the overall ensemble probability by decreasing the number of blank traces. A single channel inactivation curve was constructed by computing the relative appearance of blank traces at different holding potentials before and after photoreleasing caged ATP. As determined in dialyzed axons, the effect of phosphorylation consisted in a shift of the inactivation curve toward more positive potentials. The 20-pS channel has the same characteristics as the delayed rectifier current in activation kinetics, steady-state inactivation, and phosphorylation effects.

scholarly journals Shaker potassium channel gating. III: Evaluation of kinetic models for activation.

1994 ◽  
Vol 103 (2) ◽  
pp. 321-362 ◽  
Author(s):  
W N Zagotta ◽  
T Hoshi ◽  
R W Aldrich
Keyword(s):  
Steady State ◽  
Conformational Changes ◽  
Time Course ◽  
Single Channel ◽  
Ionic Currents ◽  
Single Step ◽  
Gating Currents ◽  
Open Probability ◽  
Shaker Potassium Channel ◽  
Single Channel Currents

Predictions of different classes of gating models involving identical conformational changes in each of four subunits were compared to the gating behavior of Shaker potassium channels without N-type inactivation. Each model was tested to see if it could simulate the voltage dependence of the steady state open probability, and the kinetics of the single-channel currents, macroscopic ionic currents and macroscopic gating currents using a single set of parameters. Activation schemes based upon four identical single-step activation processes were found to be incompatible with the experimental results, as were those involving a concerted, opening transition. A model where the opening of the channel requires two conformational changes in each of the four subunits can adequately account for the steady state and kinetic behavior of the channel. In this model, the gating in each subunit is independent except for a stabilization of the open state when all four subunits are activated, and an unstable closed conformation that the channel enters after opening. A small amount of negative cooperativity between the subunits must be added to account quantitatively for the dependence of the activation time course on holding voltage.

scholarly journals Effective Activation of BKCa Channels by QO-40 (5-(Chloromethyl)-3-(Naphthalen-1-yl)-2-(Trifluoromethyl)Pyrazolo [1,5-a]pyrimidin-7(4H)-one), Known to Be an Opener of KCNQ2/Q3 Channels

2021 ◽  
Vol 14 (5) ◽  
pp. 388
Author(s):  
Wei-Ting Chang ◽  
Sheng-Nan Wu
Keyword(s):  
Single Channel ◽  
Slow Component ◽  
Ionic Channel ◽  
Gh3 Cells ◽  
Bkca Channel ◽  
Bkca Channels ◽  
Gating Charge ◽  
Ramp Voltage ◽  
K Current ◽  
The Mean

QO-40 (5-(chloromethyl)-3-(naphthalene-1-yl)-2-(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-7(4H)-one) is a novel and selective activator of KCNQ2/KCNQ3 K+ channels. However, it remains largely unknown whether this compound can modify any other type of plasmalemmal ionic channel. The effects of QO-40 on ion channels in pituitary GH3 lactotrophs were investigated in this study. QO-40 stimulated Ca2+-activated K+ current (IK(Ca)) with an EC50 value of 2.3 μM in these cells. QO-40-stimulated IK(Ca) was attenuated by the further addition of GAL-021 or paxilline but not by linopirdine or TRAM-34. In inside-out mode, this compound added to the intracellular leaflet of the detached patches stimulated large-conductance Ca2+-activated K+ (BKCa) channels with no change in single-channel conductance; however, there was a decrease in the slow component of the mean closed time of BKCa channels. The KD value required for the QO-40-mediated decrease in the slow component at the mean closure time was 1.96 μM. This compound shifted the steady-state activation curve of BKCa channels to a less positive voltage and decreased the gating charge of the channel. The application of QO-40 also increased the hysteretic strength of BKCa channels elicited by a long-lasting isosceles-triangular ramp voltage. In HEK293T cells expressing α-hSlo, QO-40 stimulated BKCa channel activity. Overall, these findings demonstrate that QO-40 can interact directly with the BKCa channel to increase the amplitude of IK(Ca) in GH3 cells.

Effect of Prozac on whole cell ionic currents in lens and corneal epithelia

1995 ◽  
Vol 269 (1) ◽  
pp. C250-C256 ◽  
Author(s):  
J. L. Rae ◽  
A. Rich ◽  
A. C. Zamudio ◽  
O. A. Candia
Keyword(s):  
Potassium Channels ◽  
Single Channel ◽  
Ionic Currents ◽  
Current Amplitude ◽  
Human Lens ◽  
Delayed Rectifier ◽  
Open Probability ◽  
Channel Current ◽  
Single Channel Current ◽  
Ionic Conductances

Prozac (fluoxetine), a compound used therapeutically in humans to combat depression, has substantial effects on ionic conductances in rabbit corneal epithelial cells and in cultured human lens epithelium. In corneal epithelium, it reduces the current due to the large-conductance potassium channels that dominate this preparation. Its effects seem largely to decrease the open probability while leaving the single-channel current amplitude unaltered. In cultured human epithelium, currents from calcium-activated potassium channels and inward rectifiers are unaffected by Prozac. Delayed-rectifier potassium currents are reduced by Prozac in a complicated way that involves both gating and single-channel current amplitude. Fast tetrodotoxin-blockable sodium currents are also decreased by Prozac in this preparation. For all of these ion conductance effects, Prozac concentrations of 10(-5) to 10(-4) M are required. Whereas these levels are 10- to 100-fold higher than the plasma levels achieved in therapeutic use in humans, they are comparable to or less than levels needed for many other blockers of the ionic conductances studied here.

Regulation of high-conductance anion channels by G proteins and 5-HT1A receptors in CHO cells

1993 ◽  
Vol 264 (3) ◽  
pp. F490-F495 ◽  
Author(s):  
A. W. Mangel ◽  
J. R. Raymond ◽  
J. G. Fitz
Keyword(s):  
G Proteins ◽  
Pertussis Toxin ◽  
Cho Cells ◽  
Single Channel ◽  
Chinese Hamster ◽  
Channel Activity ◽  
Anion Channels ◽  
Open Probability ◽  
Single Channel Currents ◽  
High Conductance

This study addresses the mechanisms responsible for regulation of high-conductance anion channels by GTP binding proteins in Chinese hamster ovary (CHO) cells. Single-channel currents were measured in inside-out membrane patches using patch-clamp techniques. Anion-selective channels with a unitary conductance of 381 +/- 8 pS activated spontaneously in 48% of excised patches. In patches with no spontaneous channel activity, addition of GppNHp, a nonhydrolyzable analogue of GTP, activated channels in 8 of 12 studies, and in patches with spontaneous channel activity, GppNHp increased open probability in 4 of 4 experiments. In contrast, GDP beta S, a nonhydrolyzable GDP analogue, inhibited both spontaneous and GppNHp-induced channel activity. In patches without spontaneous channel activity, addition of cholera toxin activated channels in five of eight studies. Interestingly, pertussis toxin had a similar effect, activating channels in five of seven previously quiescent patches. To further evaluate the possible role of inhibitory G proteins in channel regulation, activity was measured in cell-attached patches in cells transfected with the serotonin 5-HT1A receptor, which is coupled to effector mechanisms through a pertussis toxin-sensitive G protein. Stimulation of 5-HT1A-transfected cells with the receptor agonist (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin caused a transient decrease in open probability in either standard or high-potassium solutions. In aggregate, these findings suggest that both cholera and pertussis toxin-sensitive G proteins contribute to regulation of high-conductance anion channels in CHO cells.

Rectification of muscarinic K+ current by magnesium ion in guinea pig atrial cells

1987 ◽  
Vol 253 (1) ◽  
pp. H210-H214
Author(s):  
M. Horie ◽  
H. Irisawa
Keyword(s):  
Guinea Pig ◽  
Action Potentials ◽  
Single Channel ◽  
Outward Current ◽  
Atrial Cells ◽  
Voltage Dependent ◽  
K Current ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Low K

Rectifying properties of the acetylcholine (ACh)-sensitive K+ channels were studied using a patch-clamp method in single atrial cells prepared enzymatically from adult guinea pig hearts. In the presence of micromolar concentration of nonhydrolyzable guanosine 5'-triphosphate (GTP) analogue 5'-guanylylimidodiphosphate (GppNHp) and the absence of Mg2+ at the inner surface of patch membrane [( Mg2+]i), the channel activity recovered in inside-out patch condition. The single channel conductance became ohmic between -80 and +80 mV (symmetrical 150 mM K+ solutions). The rapid relaxation of outward single channel currents was disclosed on a depolarization. [Mg2+]i blocked the outward current through the channel dose- and voltage-dependently and also induced a dose-dependent increase in the channel activation. The apparent paradoxical role of [Mg2+]i is important for the cholinergic control in the heart; voltage-dependent Mg block ensures a low K+ conductance of cell membrane at the plateau of action potentials during the exposure to ACh, thereby slowing the heart rate without unfavorable shortening of the action potentials.

scholarly journals Spermine Block of the Strong Inward Rectifier Potassium Channel Kir2.1

2002 ◽  
Vol 120 (1) ◽  
pp. 53-66 ◽  
Author(s):  
Lai-Hua Xie ◽  
Scott A. John ◽  
James N. Weiss
Keyword(s):  
Single Channel ◽  
Quantitative Model ◽  
Inward Rectification ◽  
Channel Blockers ◽  
Surface Charges ◽  
Open Probability ◽  
Dependent Component ◽  
Voltage Dependent ◽  
Single Channel Currents ◽  
Inside Out

Inward rectification in strong inward rectifiers such as Kir2.1 is attributed to voltage-dependent block by intracellular polyamines and Mg2+. Block by the polyamine spermine has a complex voltage dependence with shallow and steep components and complex concentration dependence. To understand the mechanism, we measured macroscopic Kir2.1 currents in excised inside-out giant patches from Xenopus oocytes expressing Kir2.1, and single channel currents in the inside-out patches from COS7 cells transfected with Kir2.1. We found that as spermine concentration or voltage increased, the shallow voltage-dependent component of spermine block at more negative voltages was caused by progressive reduction in the single channel current amplitude, without a decrease in open probability. We attributed this effect to spermine screening negative surface charges involving E224 and E299 near the inner vestibule of the channel, thereby reducing K ion permeation rate. This idea was further supported by experiments in which increasing ionic strength also decreased Kir2.1 single channel amplitude, and by mutagenesis experiments showing that this component of spermine block decreased when E224 and E299, but not D172, were neutralized. The steep voltage-dependent component of block at more depolarized voltages was attributed to spermine migrating deeper into the pore and causing fast open channel block. A quantitative model incorporating both features showed excellent agreement with the steady-state and kinetic data. In addition, this model accounts for previously described substate behavior induced by a variety of Kir2.1 channel blockers.

Complexity of the outward K+ current of the rat megakaryocyte

1997 ◽  
Vol 272 (5) ◽  
pp. C1525-C1531 ◽  
Author(s):  
E. Romero ◽  
R. Sullivan
Keyword(s):  
K Channel ◽  
Delayed Rectifier ◽  
Channel Blockers ◽  
Excitable Cells ◽  
Electrophysiological Properties ◽  
Relative Contribution ◽  
Delayed Rectifier Current ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
K Current

Megakaryocytes isolated from rat bone marrow express a voltage-dependent, outward K+ current with complex kinetics of activation and inactivation. We found that this current could be separated into at least two components based on differential responses to K+ channel blockers. One component, which exhibited features of the "transient" or "A-type" K+ current of excitable cells, was more strongly blocked by 4-aminopyridine (4-AP) than by tetrabutylammonium (TBA). This current, which we designated as "4-AP-sensitive" current, activated rapidly at potentials more positive than -40 mV and subsequently underwent rapid voltage-dependent inactivation. A separate current that activated slowly was blocked much more effectively by TBA than by 4-AP. This "TBA-sensitive" component, which resembled a typical delayed rectifier current, was much more resistant to voltage-dependent inactivation. The relative contribution of each of these components varied from cell to cell. The effect of charybdotoxin was similar to that of 4-AP. Our data indicate that the voltage-dependent K+ current of resting megakaryocytes is more complex than heretofore believed and support the emerging concept that megakaryocytes possess intricate electrophysiological properties.

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